15 Final steps for the preparation of the D altrose analogue 216

Scheme 3.15.Final steps for the preparation of theD-altrose analogue216.

Likewise a suitable crystal of hexose 216 was subjected to X-ray structure analysis and the resultant β-anomer is shown in Figure 3.9. Again, the structure confirms the expected stereochemistry, consistent with the anticipated stereochemical course of the various C-F bond forming reactions during the synthesis.

Figure 3.9.X-ray crystal structure of theD-altrose analogue216(β-anomer).

In both of the X-ray structures of the trifluorosugars 193 and 216, the crystal packing revealed a partitioning of the oxygens/hydroxyls from the fluoromethylene residues, an intermolecular pattern most probably imposed by the stronger hydrogen bonding interactions between the oxygen atoms and hydroxyl groups. This can be seen in Figure 3.10 for the D-glucose analogue 193 where the shortest intermolecular O--H and F--H

216 F O F F OH HO

interactions are 1.74 and 2.43 Å respectively, and in Figure 3.11 for the D-altrose analogue 216 where the shortest intermolecular O--H and F--H interactions are 1.82 and 2.42 Å respectively.

Figure 3.10.Molecular packing of analogue193(β-anomer).

Figure 3.11.Molecular packing of analogue216(β-anomer).

O/OH interface CH/CF interface O/OH interface CH/CF interface

102

3.3.1.5) NMR analysis ofD-glucose analogue 193andD-altrose analogue216:

NMR analysis provides19F chemical shifts as well as19F-1H,19F-13C and19F-19F coupling constants as parameters to obtain structural information.6 However, the presence of three vicinal fluorines in 193 and 216 generated a complex 1D spectrum. As a result, an unambigous assignment of all the 1H, 13C and 19F signals was not possible due to extensive overlap of the NMR resonances. In these cases, carrying out 2D NMR analyses such as 1H-19F HMBC and 19F-19F COSY as well as 1D analysis such as 1H{19F}-NMR, was essential to assist confident signal assignments.

In order to assign the specific anomers in the CDCl3 solutions of 193and 216, a series of

1D and 2D NMR experiments was carried out. The 1H-NMR spectrum of 193 (Figure 3.12) shows two doublet of doublets assigned to H1 (α or β), one at 5.48 ppm displaying coupling constants of 3.7 and 3.1 Hz, and one at 4.91 ppm displaying coupling constants of 7.6 and 3.1 Hz. The 1H{19F}-NMR spectrum of 193 in CDCl3 allowed discrimination

between the 3JH-H and 3JH-F coupling constants. The spectrum (Figure 3.13) shows two

doublets at 5.48 ppm and 4.91 ppm displaying3JH-H coupling constants of 3.7 and 7.6 Hz

respectively. The larger 3JH-H coupling constant of 7.6 Hz is indicative of a trans-diaxial

relationship between the protons H1 and H2, thus the signal at 4.91 ppm was assigned to the β-anomer. By inference, the signal at 5.48 ppm was assigned to the α-anomer. Integration of these signals indicated that the anomers were present in a ratio of 1:0.2 (α:β) (Figure 3.13).

Figure 3.12.1H-NMR spectrum of193in CDCl3.

Figure 3.13.1H{19F}-NMR spectrum of193in CDCl3.

H1α

104

The 1H,19F-HMBC spectrum of 193 in CDCl3 allowed an assignment of F2α and F2β. The

spectrum (Figure 3.14) shows 3JH1α-F2α and 3JH1β-F2β crosspeaks which enabled an

assignment of the doublet of doublets at -199.9 ppm and -200.5 ppm to F2α and F2β, respectively.

Figure 3.14.1H,19F-HMBC spectrum of193in CDCl3.

The19F{1H}-NMR spectrum of193in CDCl3(Figure 3.15) allowed assignment of F3α/F3β

and F4α/F4βto the corresponding signals. The spectrum shows six signals corresponding to the α- and β- anomers. The two doublet of doublets at -199.9 ppm and -200.5 ppm had previously been assigned to F2α and F2β, respectively (Figure 3.14). The signals at -195.8 ppm and -201.6 ppm are triplets whereas the signals at -201.2 ppm and -202.0 ppm are doublet of doublets (Figure 3.15). These two triplets arise due to the similar 3JF3-F2 and 3

JF3-F4 coupling constants and are thus assigned to F3 (α or β) (Figure 3.15). Furthermore

integration of these signals gave a ratio of 1:0.2, corresponding to the ratio of the α- and

β- anomers respectively (Figure 3.15). By inference, the doublet of doublets at -202.0

H1α

H1β

F2β F2α

ppm was assigned to F4αand the doublet of doublets at -201.2 ppm to F4β (Figure 3.15). The anomeric ratio of 193 tends towards 1:1 by increasing the polarity of the solvent (Figure 3.16), furthermore a significant change in the19F- chemical shifts are observed.

Figure 3.15.19F{1H}-NMR spectrum of193in CDCl3.

Figure 3.16.19F{1H}-NMR showing the α/β anomer ratios of193 in (a) CDCl3(α:β, 1:0.13); (b) CD3CN

(α:β, 1:1); (c)d8-THF (α:β, 1:1). F3β F3α F2α F2β F4β F4α (a) (b) (c) β α β β α α

106

The 1H-NMR spectrum (Figure 3.17, section a) of 216 in CDCl3 shows a doublet of

doublets at 5.32 ppm, displaying both large and small coupling constants of 11.1 and 1.6 Hz, allowing assignment to H1 (α or β). Although the accurate measurement of the coupling constants for the resonance at 5.19 ppm (assigned to the other anomeric proton H1) was not achievable due to overlapping of signals, a large coupling constant of approximately 17 Hz was estimated. This coupling constant is lost in the 1H{19F}-NMR spectrum (Figure 3.17, section b), thus the larger coupling constant of 17 Hz is indicative of atrans-diaxial relationship between H1βand F2β, whereas the smaller coupling constant (11.1 Hz) is indicative of an axial-equatorial relationship between H1α and F2α. Thus the signals at 5.32 and 5.19 ppm were assigned to H1α and H1β, respectively. Also, the integration of these signals shows a ratio of 0.8:1 corresponding to the ratio of the α- and

β- anomers respectively.

Figure 3.17.(a)1H and (b)1H{19F}-NMR of216in CDCl3.

A 19F{1H}-NMR spectrum of 216 in CDCl3 was recorded (Figure 3.18) and showed six

signals corresponding to the α- and β- anomers. Integration (α:β ratio of 0.8:1) enabled assignment of the resonances at -200.5, -209.7 and -213.8 ppm to the α-anomer, and of

b)

a)

H1α H

the resonances at -213.4, -214.3 and -219.3 ppm to the β-anomer. However, an unambigous assignment of these resonances to the corresponding fluorines (F1α and F1β, F2αand F2β, F3αand F3β) by1H,19F-HMBC was not possible.

Figure 3.18.19F{1H}-NMR spectrum of216in CDCl3.

3.3.2) Assessment of the erythrocyte transmembrane transport ofD-glucose analogue

193 and D-altrose analogue 216:

The ability of the trifluoro D-hexose analogues 193 and 216 to cross erythrocyte membranes was evaluated. 19F-NMR and 2D 19F EXSY-NMR experiments were carried out to determine the efflux rate constants (kef) from human erythrocyte suspended in a

D2O buffer solution. Because of the different anomeric permeability across the

erythrocyte membrane, the assignment of the specific anomers to 193 and 216 in a deuterated buffer (D2O-Tris-HEPES) was first required. To achieve this, a series of 1D

α α

α β

β

108

and 2D NMR experiments was carried out. The 1H{19F}-NMR analysis of 193 in D2O

buffer allowed assignment of H1 in each anomer. The spectrum (Figure 3.19) shows two doublets assigned to H1 (α or β), one at 5.50 ppm displaying a 3JH-H coupling constant of

3.8 Hz, and one at 5.00 ppm displaying coupling constant of 8.2 Hz. The larger 3JH-H

coupling constant is indicative of a trans-diaxial relationship between the H1 and H2 protons, thus the signals at 5.00 ppm and 5.50 ppm were assigned to the β- and α- anomers, respectively. Integration of these signals indicated that the anomers are present in 1:0.9 ratio (α:β).

Figure 3.19.1H{19F}-NMR spectrum of193in D2O-Tris-HEPES buffer.

The 1H,19F-HMBC spectrum of 193 in D2O-Tris-HEPES buffer in Figure 3.20 shows 3

JH1α-F2 and 3JH1β-F2 crosspeaks which enabled assignment of the doublets at -199.4 ppm

and -200.7 ppm to F2αand F2β, respectively.

Figure 3.20.1H,19F-HMBC spectrum of193in D2O-Tris-HEPES recorded at 37 °C.

The use of 1H-NMR (Figure 3.21, section a) and 1H{19F}-NMR (Figure 3.21, section b) spectra to assign H1 to the corresponding anomers (α or β) of 216 in D2O-Tris-HEPES

was not achievable, due to overlap of H1(α andβ) and H3(Figure 3.21). To overcome this obstacle, the 1D gradient NOESY spectra was recorded in D2O-Tris-HEPES buffer

(Figure 3.22). The spectrum shows an NOE between H5 (4.39 ppm) and a H6 proton (Figure 3.22, section a), and also between H5 (4.13 ppm) and both H6 and H1 protons (Figure 3.22, section b). This enabled assignment of the resonance at 4.39 ppm to H5and that at 4.13 ppm to H5.

Figure 3.21.(a)1H-NMR and (b)1H{19F}-NMR spectra of216in D2O-Tris-HEPES.

H1 α H1β F2α F2β H1b H1a H2+ H4 H5 a) b) H6 H1+ H3

110

Figure 3.22. 1D gradient NOESY spectra of216 in D2O-Tris-HEPES: spectrum (a) shows NOE between

H5at 4.39 ppm and H6; spectrum (b) shows NOE between H5at 4.13 ppm and H6and H1.

The 1H,19F-HMBC spectrum of 216 in D2O-Tris-HEPES buffer was recorded in order to

assign F2, F3 and F4 to the corresponding anomers. The spectrum (Figure 3.23) shows

3

JH5α-F4α,4JH5α-F3α crosspeaks which enabled an assignment of the signals at -212.0 ppm

and -207.3 ppm to F3α and F4α respectively, and 3JH5β-F4β and4JH5β-F3β(-214.3 ppm and -

213.2 ppm) to F3β and F4β respectively. F2 resonances (α and β) were assigned using

19

F,19F-COSY (not shown). a) b) H5 H5 H6 H6 H1 O F F F OH H1 -anomer OH H5 H6

Figure 3.23.1H,19F-HMBC spectrum of216in D2O-Tris-HEPES buffer.

The next step of the erythrocyte transmembrane transport study required the preparation of a suspension of 193 and red blood cells in D2O buffer. The experiment with 216 was

performed in parallel. Human red blood cells were obtained from First Link (UK) Ltd. The human blood was centrifuged (4000 rpm, 5 min) and the plasma and supernatant were discarded. The cells were then washed four times in three volumes of saline buffer solution containing 123 mM NaCl, 15 mM Tris-HEPES and 5 mM ascorbic acid (previously filtered through sterile filters Nalgene 0.20 µm cellulose acetate membranes), and after each wash the supernatant was removed and the cells were collected by centrifugation. The red blood cells were then transferred to an Eppendorf tube and diluted with one volume of saline buffer and 20 mM of the trifluoroglucose analogue 193 (final hematocrit 0.5), carefully mixed and transferred into a WILMAD NMR tube with a J Joung valve. A sealed capillary tube containing D2O was then introduced into the NMR

H5α H5β F3β F4β F3α F4α F2α F2β

112

tube to provide a deuterium lock signal. Also a stream of carbon monoxide was bubbled through the cells for 3 minutes before sealing in order to eliminate any paramagnetic deoxyhemoglobin which can broaden the NMR resonances. The sample was sealed and was ready to be analysed by 2D 19F EXSY-NMR spectroscopy. A control experiment consisted of the 1F{1H}-NMR spectra of 193 in D2O-Tris-HEPES buffer, recorded at 37

°C. This showed six resonances (Figure 3.24, section a) corresponding to the α- and β- anomers as previously assigned.

The19F EXSY-NMR spectrum (consisting of two 19F{1H}-NMR spectra perpendicular to each other) of 193, in the presence of erythrocytes suspended in the buffer at 37 °C showed twelve resonances corresponding to the intra- and extra- cellular populations of the - and - anomers (Figure 3.24, section b). All six resonances of the anomers of 193

were associated with broad downfield shifted resonances corresponding to the intracellular population of 193. Furthermore the 19F EXSY-NMR spectrum shows cross- peaks indicative of an exchange between the intra- and extra- cellular pools. Specifically, the intensities of the selected cross-peaks (Figure 3.24, section b) associated with F2α (signal at -199.3 ppm) and F3β (signal at -195.7 ppm) are proportional to the exchange rates of the - and - anomers between the intra- and extra- cellular enviroments. The efflux rate constants (kef) (Table 3.3) were calculated importing the quantified EXSY data

Figure 3.24 (a) 19F{1H}-NMR spectrum (470.4 MHz) (control experiment) of 193 in D2O-Tris-HEPES

buffer (b)19F EXSY-NMR spectrum of193in the presence of erythrocytes suspended in the buffer at 37 °C.

Over time a new metabolite appeared (Figure 3.25). Although the identification of this metabolite was not successful, it is apparent from the 19F{1H}-NMR spectrum that the metabolite is generated as a single diastereoisomer, and this might be indicative of an oxidation at the anomeric position to generate 204 (Figure 3.26). The formation of this metabolite was very slow, and did not interfere with the determination of the exchange rates of193. a) b) F2α F3β F2β/F3α F4α F4β

114

Figure 3.25. 19F{1H}-NMR spectrum (470.4 MHz) of 193 in the presence of erythrocytes suspended in D2O-Tris-HEPES buffer at 37 °C, showed additional signals corresponding to metabolite formation.

O F F F OH 204 O

Figure 3.26.Putative metabolite generated by oxidation of193at the anomeric position.

London et al.8 evaluated the human erythrocyte transmembrane transport of the 3-deoxy- 3-fluoro-D-glucose164, and showed transport ability similar to that of the D-glucose. The corresponding kef is reported in Table 3.4. This experiment was now repeated, and the

resultingkef(Table 3.4) found to be in good agreement with the literature.8The D-glucose 193 and D-altrose 216 analogues cross the human erythtrocyte membrane less efficiently when compared to 164, but 193 was transported better (~70% efficiency) than 216.

Additional signals corresponding to metabolite

Furthermore, for 193there is a marked preference for transport of the α-anomer over the

β-anomer. Conversely, for 216, transport is slow and there is no obvious preference for a particular anomer. kef [s-1] -anomer -anomer 164(3-F-D-glucose)a 1.35±0.32 1.04±0.23 164(3-F-D-glucose)b 1.38±0.02 1.01±0.08 193(D-glucose)b 0.97±0.21 0.22±0.07 216(D-altrose)b 0.33±0.07 0.40±0.05

a_{Data from the literature.}8 b_{Data reproduced in this work.}

Table 3.4.Efflux rate constants for164,193and 216across erythrocyte cell membranes measured by 2D

19

F EXSY-NMR. The values reported are averages from different 19F resonances and mixing times. Errors are standard deviations.